In normal female meiosis the precursor cells of the ova multiply and then reduce the number of chromosomes to one half set in each gamete in two specialised meiotic divisions.
Meiosis is initiated in the fetal ovary before birth during the early development of the female germ cells (primary oocytes), which will eventually form mature eggs (or oocytes, the terms are used interchangeably) in the adult female.
To reduce the number of chromosomes from the normal (euploid) 23 pairs of homologous chromosomes (one of each pair inherited from the father and one from the mother, so 46 in total) to 23 single chromosomes, there is one round of DNA replication in which each chromosome is duplicated into two sister chromatids followed by two specialised meiotic divisions, meiosis I and II.
Following replication, the two homologous chromosomes of each pair ‘pair up’ and a single bivalent chromosome forms in which all four sister chromatids are tightly bound together. This allows a limited number of breaks in the DNA strands of adjacent non-sister chromatids to ‘crossover’ and re-join the other chromatid, leading to non-recombinant (no exchange) and recombinant chromatids and generating genetic variation.
As the cell divides at the end of meiosis I, one homologous chromosome of each pair is pulled into the first polar body and the other into the secondary oocyte, which therefore now has 23 chromosomes each with two sister chromatids.
In meiosis II, following fertilisation of the oocyte by a sperm cell containing the paternal half set of chromosomes, the two sister chromatids of each chromosome finally separate and segregate into the second polar body and the fertilised oocyte (now a zygote). The zygote therefore inherits 23 single maternal chromatids (now ‘chromosomes’).
Aneuploidy is defined as an abnormal number of whole chromosomes or parts of chromosomes causing a genetic imbalance. The most frequent and clinically significant aneuploidies involve single chromosomes (strictly ‘aneusomy’) in which there are either three (‘trison’) or only one (‘monosomy’) instead of the normal pair of chromosomes per somatic cell.
Chromosome aneuploidy is a major cause of pregnancy loss and abnormal pregnancy with live births and increases exponentially with maternal age in the decade preceding menopause (Hassold and Hunt, 2001). Most autosomal aneuploidies and all autosomal monosomies are lethal, only a small number of trisomies are compatible with full term development often with severe congenital abnormalities.
A similar pattern of aneuploidy occurs in pregnancies following assisted conception using in vitro fertilisation (IVF) (Spandorfer et al., 2004). Furthermore, microarray based comparative genomic hybridisation (e.g., array CGH) analysis has shown that a majority of human oocytes in women of advanced maternal age (average age 40) are aneuploid, most with multiple aneuploidies (Handyside et al., 2012).
Currently, human oocytes can be tested for aneuploidy using whole genome amplification (WGA) of both the first and second polar bodies (PB1 and PB2, respectively) by microarray based comparative genomic hybridisation (array CGH). Array CGH is a methodology, which compares the amount of DNA hybridising to DNA probes spaced typically at 1 Mb intervals across the genome, i.e. across each chromosome, in test and control DNA labelled with green and red fluorochromes (24Sure™, BlueGnome Ltd; www.24Suretest.com), for example. With human oocytes, WGA products from the two polar bodies are labelled and hybridised and the signal intensities compared to control male and female DNA labelled in opposite fluorochromes.
The polar bodies are by-products of the two meiotic divisions, meiosis I and II, and since they do not form part of the embryo they can be removed with minimal effect using published methods well known to those skilled in the art. However, biopsying both polar bodies from each oocyte is labour intensive for clinics and multiple arrays are required to test each oocyte.
Other methods for detecting aneuploidy have been proposed as described herein.
For example, the presence or absence of each chromosome in polar bodies can be detected by multiplex PCR of panels of chromosome specific sequences (Advalytix, Beckman Coulter; www.advalytix.com/advalytix/). However amplification bias makes it difficult to accurately quantify the products, thereby limiting possible application for aneuploidy testing in polar bodies.
Limiting dilution into separate wells and digital PCR can be used to count the number of chromatids. However by virtue of the steps involved, this methodology can be technically challenging, and has not yet been extensively validated (for example, see publication WO2011/138750 of the MRC et al).
Thus it can be seen that novel, less complex, methods for assessing the risk of aneuploidy of eggs, fertilised eggs or embryos developed therefrom would provide a contribution to the art.